Vaccinia virus is used to vaccinate persons to prevent disease with smallpox. Compared with other live virus vaccines, the smallpox vaccine is associated with frequent side effects including fever, a sore or swollen arm, headache, and fatigue. Other more serious side effects have also been reported including development of multiple lesions outside the vaccination site (generalized vaccinia), inflammation of the brain (postvaccinia encephalitis), severe infection of the skin in persons with a history of eczema (eczema vaccinatum), and disseminated vaccinia virus infection in immunocompromised persons (progressive vaccinia). Relatively few studies of vaccine-induced immune responses have been done in the modern era using contemporary techniques. Cytokines are proteins that are produced by white blood cells and secreted into the bloodstream that are important for trafficking of white blood cells in response to inflammation. We hypothesize that induction of cytokines is associated with some of the side effects of smallpox vaccination. Identification of specific cytokines induced after vaccination may help to explain the pathogenesis of certain side effects associated with smallpox vaccination and might suggest new ways to modify some of these side effects. The diagnosis of complications related to smallpox vaccination may be difficult because the rash may resemble other infectious or noninfectious diseases. We have analyzed three different methods to detect the smallpox vaccine virus in specimens from patients who were vaccinated. We compared a cell culture assay that detects infectious virus, a polymerase chain reaction (PCR) test that detects virus DNA, and a direct fluorescent antibody test (DFA) that detects virus proteins. We found that our PCR test detected the smallpox vaccine virus as well as other members of the same class of viruses (orthopoxviruses). All of the commercially available antibodies used for the antibody test (DFA) detected the smallpox vaccine virus, other poxviruses, and surprisingly herpes simplex virus type-1. The PCR test was the most sensitive (100% of specimens positive), the cell culture test was the next most sensitive test (93% of specimens positive), while antibody test (DFA) was the least sensitive (27% of specimens positive). The antibody test was the fastest test to perform, but was limited by adequate numbers of cells in specimens. The PCR test was positive for a longer time post-vaccination than the cell culture test.